Activated peptides and conjugates

ABSTRACT

Novel activated peptides and conjugates thereof, useful in diagnostic assays and therapeutics, and processes for the preparation thereof are disclosed.

This is a division of Ser. No. 08/833,546 filed Apr. 7, 1977, now U.S.Pat. No. 5,977,299.

The present invention relates to activated peptides, conjugates andmethods of preparation thereof, and their use in diagnostic assays andtherapeutics.

Numerous assays have been developed for the detection and determinationof proteins, e.g., antibodies in biological fluids. One class, theimmunoassays, which has evolved into an invaluable tool in diagnostics,is based upon the principle of specific binding of antibodies to haptensand/or antigens. In a typical immunoassay, a test sample, containing ananalyte of interest, and an antibody, to which it specifically binds,are incubated, and then washed to separate free and bound analytes. Anenzyme-labeled antibody that recognizes the resulting complex is added,incubated, and washed, and finally substrate, an enzyme detectionsystem, is added and the labeled complex is detected and determined.

Conjugates of peptides specific to antibodies have been usedadvantageously in immunoassays. Conjugates of peptide analogues of viralproteins, that is, segments of the proteins bearing the epitopicsequence, for example, of the human immunodeficiency viruses (HIV) andlabeled enzymes linked through a maleiimide moiety have been describedas being particularly advantageous in the detection and determination ofantibodies to HIV. See U.S. Pat. No. 5,294,536 granted to Paul S.Palumbo on Mar. 15, 1994 ('536-patent). The conjugates of activatedpeptides, prepared by the processes described in the '536-patent are nothomogenous, the activated peptides being derived from the terminal aminogroup and/or the internal amino and hydroxyl groups of the peptideanalogue. Interaction of the internal amino and/or hydroxyl groups ofthe peptide analogue and the crosslinking agent, in addition to that ofthe terminal amino group, diminishes the effectiveness of the epitopiccenters of the peptide analogue thereby reducing the sensitivity of theassay for the detection and determination of specific bindingantibodies. For example, interaction of the amino group of the lysinesubunit and/or the amino and the hydroxy subunits of the serine subunitof the epotopic segment of the peptide analog of the HIV virus and thecrosslinker reduces the sensitivity of the immunoassay for the detectionor determination of antibodies to HIV, the greater the interaction ofthe internal amino and/or hydroxyl group relative to the terminal aminogroup, the lower the sensitivity of the assay. The processes for thepreparation of activated peptides 3 and conjugates 5 described in theaforementioned '536-patent involve condensation of anisocyanatomaleiimide 1

wherein X is a spacer group with a peptide having a terminal amino groupof formula 2

RNH₂  2

wherein R is the remainder of the peptide to form an activated peptide 3

wherein R and X are as above, which is then condensed with a thiolatedenzyme of formula 4

R₁SH  4

wherein R₁ is the remainder of an enzyme to form a conjugate of formula5

wherein X, R, and R₁ are as defined herein. The critical step in theprocess, the reaction of the crosslinker 1 with a peptide 2,characterized by the presence of a terminal amino group and internalamino and/or hydroxyl groups, affords an activated peptide 3 derivedfrom the terminal amino group, as well as activated peptides derivedfrom the internal amino and/or hydroxyl groups of the peptide, andcombinations thereof. It has now been found that by performing thereaction of the isocyanatomaleiimide 1 with a peptide 2 as a salt of astrong protonic acid, the activated peptide is formed substantially freeof activated peptides derived from internal amino and/or hydroxylgroups, e.g., of serine or lysine, i.e., the reaction takes place almostexclusively at the terminal amino group of the peptide 2 toregiospecifically form an activated peptide 3 with the epitopic segmentof the peptide 2 essentially intact.

It has now also been found that the integrity of the binding region ofthe activated peptide 3 is maintained in the conjugate 5 and that use ofthe essentially homogeneous conjugate 5 of the invention in animmunoassay results in a marked improvement of the sensitivity of theassay.

More particularly, the present invention relates to activated peptidesof formula 3

wherein X is loweralkylene, an aromatic carbocyclic moiety or asaturated carbocyclic moiety and R is the remainder of a peptide havinga terminal primary amino group and free internal hydroxyl and/or aminogroups, useful for the preparation of conjugates 5

wherein R and X are as defined above and R₁ is the remainder of anenzyme having a free thiol group, useful for the detection anddetermination of antibodies in samples of interest related to the humanimmunodeficiency viruses.

Preferred activated peptides 3 are those wherein X is an aromaticmoiety; most preferred are peptides wherein X is phenyl, and R is theremainder of a peptide analogue having a terminal amino group selectedfrom the group consisting of

As used throughout the specification and appended claims, the termalkylene refers to a saturated straight or branched chain hydrocarbonhaving 1 to 10 carbon atoms. Examples of alkylene groups are methylene(—CH₂—), ethylene (—CH₂CH₂—), butylene

The term “lower” as applied to alkylene groups refers to a carbonskeleton having 1 to 6 carbon atoms.

The term “alpha-amino acid” refers to a compound characterized by thepresence of a carboxylic acid group and an alpha-amino group bound tothe same carbon atom

Examples of amino acids are alanine, valine, leucine, isoleucine,proline (or hydroxyproline), phenylalanine, tryptophan, methionine,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, and histidine.

The term “peptide” refers to polymers of two or more amino acids linkedcovalently through the carboxyl group of an amino acid and the aminogroup of another with the elimination of water. Examples of peptides arethose compiled herein in the Sequence Listing.

The term “protein” refers to a macromolecule of one or more chains ofamino acids bound covalently through peptide bonds

Proteins include enzymes, antibodies, antigens, peptides and the like.

The term “hapten” refers to a low-molecular-weight compound that reactsspecifically with an antibody, but does not stimulate antibodyproduction unless complexed with a carrier protein.

The term “antigen” refers to a substance or entity (usually a protein)that induces the direct production of antibodies.

The term “spacer group” refers to a moiety bridging the isocyanato andmaleiimides groups and linking the protein and thiolated enzyme moietiesof a conjugate. Examples of spacer groups are alkylene (hereindefined),aromatic carbocyclics, such as phenyl or naphthyl optionally having analkylene group for attachment to the maleiimide moiety anddimethylamino, methoxy, ethoxy, methyl, ethyl, sulfonamido, sulfonicacid substituents, and saturated carbocyclics such as cyclohexyl,cyclohexylalkyl, cyclopentyl, cyclopentylalkyl, cycloheptyl,cycloheptylalkyl.

The term “regiospecific” refers to a process in which one specificstructural or positional isomer is formed to the essential exclusion ofother possible isomers.

The term “remainder” as applied to peptides refers to the moiety boundto the terminal amino group thereof, for example, the terminal aminogroup of the terminal glycine moiety of the sequence of amino acidsshown below:

The term “remainder” as applied to an enzyme having a free thiol grouprefers to the moiety bound to the thiol group thereof, for example,specific enzymes such as beta-D-galactosidase, peroxidase, glucoseoxidase and alkaline phosphatase, in which a thiol group has beenintroduced.

The N-terminal activated peptides 3 of the present invention areprepared by contacting an isocyanatomaleiimide 1 with a peptide 2 as asalt of a strong protonic acid, having in the remainder free aminoand/or hydroxyl groups capable of interaction with the isocyanato moietyof the crosslinker 1, in a suitable solvent, conditions under which theterminal amino group regioselectively reacts with the isocyanato moietyof the crosslinker 1, without substantial interaction of the internalamino and/or hydroxyl groups with the isocyanato moiety. Included amongstrong protonic acids are hydrohalic acids such as hydrobromic acid andhydrochloric acid. Also included among such acids are haloacetic acids,for example, trichloroacetic and trifluoroacetic acid. Haloacetic acidsare preferred. Trifluoroacetic acid is most preferred.

A variety of peptides 2 containing a terminal amino moiety arecommercially available, many as salts of trifluoroacetic acid. In theevent the peptide 2 is not available as the trifluoroacetic acid salt,hydrohalic and haloacetic acid salts may be prepared by conventionalmethods.

Suitable solvents include dipolar aprotic solvents, for example,dimethylformamide, dimethylacetamide, hexamethylphosphoramide, ordimethylsulfoxide. Dimethylformamide is the preferred solvent.

The reaction of a terminal amino group of a peptide 2 with a crosslinker1 is carried out at a temperature compatible with the stability of thereactants, generally as determined by the stability of the peptide 2.Typically, the reaction is performed at about 30° C.

The relative amounts of the peptide 2 and crosslinker 1 are notcritical. With about a three-fold excess of crosslinker 1 to peptide 2,good yields of the activated peptide 3 are obtained.

The reaction of a peptide 2 and crosslinker 1 is worked up byconventional methods and the product is purified by knownchromatographic techniques, for example, reverse phase high performanceliquid chromatography.

The activated peptide 2 was analyzed by various spectral techniques, forexample, mass spectrometry; and the regiospecifity of reaction of thecrosslinker 1 with the N-terminal amino group of the peptide 2 wasestablished by aminopeptidase and trypsin digestion in combination withmass spectral analyses. See, for example, K. Arar, et al., TetrahedronLetters, 34, 8087 (1993), ref 15, and B. Keil in The Enzymes, P. D.Boyer, Editor, Vol. 111, Academic Press, New York, N.Y., 1971, Chapter8, for a discussion of these methods of determining the site of reactionof a peptide having multiple amino and/or hydroxyl groups with acrosslinker such as an isocyanatomaleiimide 1.

The yields of the activated peptide 3 of the present invention preparedby the reaction of an isocyanatomaleiimide 1 with a peptide 2 areuniformly high, the crude yields by chromatographic techniques fallingwithin the range of about 49 to about 91%, the isolable yields fallingwithin the range of about 47 to about 60%.

The purity of the activated peptides 3, the condensation products ofisocyanatomaleiimides 1 and N-terminal peptides 2, was established byreverse phase high performance liquid chromatography.

The conjugates 5 of the present invention are useful in enzyme-linkedimmunoassays (ELISA) for the detection and determination of proteins,e.g., antibodies, particularly antibodies to human immunodeficiencyviruses in a ELISA module of the OPUS® system. See H. J. Crowley and M.A. Bandin in The Immunoassay Handbook, D. Wild, Edition, Stockton Press,New York, N.Y., 1994, page 197. In such an assay, e.g., an analyte ofinterest of a biological sample, e.g., an antibody, and the conjugateare incubated, applied to antibody capturing support, washed with asubstrate, incubated and detected. In particular, the sample of interestcontaining an antibody to a human immunodeficiency virus and theconjugate of a peptide derived from a glycoprotein of a humanimmunodeficiency virus are incubated, applied to a matrix of a fusionprotein, washed with methyl umbelliferyl phosphate, incubated, and theamount of conjugate bound to the peptide specific antibody is detectedand determined by fluorimetry.

Enzymes that contain a thiol group such as β-D-galactosidase and thosethat do not such as peroxidase, glucose oxidase and alkalinephosphatase, into which a thiol may be introduced, can be employed inthe conjugation with activated peptides.

The activated peptides 3 of the present invention are also useful astherapeutics for the treatment of disease. See K. Arar, ibid., page 1and references cited thereon, as well as Gary A. Koppel, BioconjugateChemistry, 1, 13 (1990).

The peptide starting materials are available from commercial sources.

The invention will now be further described with respect to specificpreferred embodiments by way of examples, it being understood that theseare intended to be illustrative only and the invention is not limited tothe materials, processes, etc., recited therein.

EXAMPLE 1

N-Terminal Activation of Peptide BC202 (Seq ID No: 2) withp-Maleiimidebenzene Isocyanate

Synthetic peptide BC202 (Seq ID No: 2), consisting of 36 amino acidswith an internal disulfide loop is derived from HIV-2 genome and is aportion of the envelope protein gp36. An N-terminal arginine, internallysine and serine, and a C-terminal serine are potential sites foractivation with p-maleiimidebenzene isocyanate (PMBI). In this example,7.1 mg of the triflororacetic acid salt of BC202 was dissolved in 1.42ml of dry dimethylformamide. To the solution a 3-molar excess (1.09 mg)of p-maleiimidebenzene isocyanate in 0.109 ml of dimethylformamide wasadded. The reaction mixture was incubated for 30 mins at 30° C.,incubated with 4.59 ml of deionized water for 5 mins at 30° C., andcentrifuged. The precipitate was collected, and the mother liquor waspurified by reverse phase high performance liquid chromatography with alinear gradient of 30%-35% acetonitrile in 0.06% trifluoroacetic over 30mins. The flow rate was 4.7 ml/min and the separation monitored at 220nm and 280 nm. The appropriate fractions were collected and evaporated.The residue was dried to yield the activated peptide as a powder; yield94% by high performance liquid chromatography, 60% by isolation.

The activated peptide was analyzed by mass spectrometry, aminopeptidasedigestion, trypsin digestion in combination with mass spectrometry, backtitration with mercaptoethanol and reaction with5,5′-dithiobis(2-nitrobenzoic acid) and conjugated to alkalinephosphatase for functional testing in an HIV-2 assay.

The reaction and separation conditions for the preparation of theactivated peptide of the invention, prepared by processes substantiallysimilar to those as described in the example, and the yields of theproducts so obtained are summarized in the Table.

TABLE Activated Peptide Separation % Yield PMBI Conditions and by HPLCPeptide* (molar ratio) Eluent** (Isolation Yield) D-23-N (SEQ ID  3XIsocratic 30% B ND NO:7) D-24-N (SEQ ID  3X Isocratic 30% B 90 NO:3)N-23-N (SEQ ID  3X Isocratic 30% B 86% (50%) NO:4) BC80 (SEQ ID 10XIsocratic 30% B 89% (60%) NO:1) BC80 (SEQ ID  3X Isocratic 30% B 77%NO:1) BCH132 (SEQ ID  3X Gradient 15-30% B 47% NO:9) 30 min. BCH408 (SEQID  3X Gradient 30-40% B 91% NO:6) 30 min. BCH178 (SEQ ID 20X Isocratic25% B 87% NO:8) BC202 (SEQ ID  3X Gradient 30-35% B 94% (60%) NO:2) 30min. BCH87 (SEQ ID  3X Gradient 32-37% B 74% (49%) NO:5) 30 min. (%)Represents isolation yields after pooling and drying. Each value is amean of two runs. *Source D-23-N (SEQ ID NO:7), Neosystems S.A.,Strasbourg, France; D-24-N (SEQ ID NO:3), Neosystems S.A., Strasbourg,France; N-23-N (SEQ ID NO:4), Neosystems S.A., Strasbourg, France; BC 80(SEQ ID NO:1), BioChem Immunosystems Inc., Montreal, Canada; BCH 132(SEQ ID NO:9), BioChem Immunosystems Inc., Montreal, Canada; BCH 408(SEQ ID NO:6), BioChem Immunosystems Inc., Montreal, Canada; BCH 178(SEQ ID NO:8), BioChem Immunosystems Inc., Montreal, Canada; BC 202 (SEQID NO:2), BioChem linmunosystems Inc., Montreal, Canada; and BCH 87 (SEQID NO:5), BioChem Immunosystems Inc., Montreal, Canada. **ChromatographyEluent 30% B-a solution of 30% by volume of a solution of 0.06% byvolume of trifluoroacetic acid in acetonitrile and 70% by volume of asolution of 0.06% of trifluoroacetic acid in deionized water. 15% B-asolution of 15% by volume of a solution of 0.06% by volume oftrifluoroacetic acid in acetonitrile and 85% by volume of a solution of0.06% of trifluoroacetic acid in deionized water. 25% B-a solution of25% by volume of a solution of 0.06% by volume of trifluoroacetic acidin acetonitrile and 75% by volume of a solution of 0.06% oftrifluoroacetic acid in deionized water. 32% B-a solution of 32% byvolume of a solution of 0.06% by volume of trifluoroacetic acid inacetonitrile and 68% by volume of a solution of 0.06% of trifluoroaceticacid in deionized water. 35% B-a solution of 35% by volume of a solutionof 0.06% by volume of trifluoroacetic acid in acetonitrile and 65% byvolume of a solution of 0.06% of trifluoroacetic acid in deionizedwater. 37% B-a solution of 37% by volume of a solution of 0.06% byvolume of trifluoroacetic acid in acetonitrile and 63% by volume of asolution of 0.06% of trifluoroacetic acid in deionized water. 40% B-asolution of 40% by volume of a solution of 0.06% by volume oftrifluoroacetic acid in acetonitrile and 60% by volume of a solution of0.06% of trifluoroacetic acid in deionized water.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 17 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:GLY CYS SER GLY LYS LEU ILE CYS THR THR AL #A VAL PRO TRP ASN ALA1               5    #                10   #                15 SER(2) INFORMATION FOR SEQ ID NO: 2:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 36 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:ARG VAL THR ALA ILE GLU LYS TYR LEU GLN AS #P GLN ALA ARG LEU ASN1               5    #                10   #                15SER TRP GLY CYS ALA PHE ARG GLN VAL CYS HI #S THR THR VAL PRO TRP            20       #            25       #            30VAL ASN ASP SER         35 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:ASP GLN ALA ARG LEU ASN SER TRP GLY CYS AL #A PHE ARG GLN VAL CYS HIS1               5    #                10   #                15THR THR VAL PRO TRP VAL ASN         20 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 23 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:ASN GLN GLN ARG LEU ASN LEU TRP GLY CYS LY #S GLY LYS LEU ILE CYS1               5    #                10   #                15TYR THR SER VAL LYS TRP ASN             20(2) INFORMATION FOR SEQ ID NO: 5:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 35 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:ARG ILE LEU ALA VAL GLU ARG TYR LEU LYS AS #P GLN GLN LEU LEU GLY1               5    #                10   #                15ILE TRP GLY CYS SER GLY LYS LEU ILE CYS TH #R THR ALA VAL PRO TRP            20       #            25       #            30 ASN ALA SER        35 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 39 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:LYS ILE LEU ALA VAL  GLU ARG TYR LEU LYS  #ASP GLN GLN LEU LEU GLY1               5    #                 10  #                 15ILE TRP  GLY CYS SER GLY LYS LEU ILE CYS  #THR THR ALA VAL PRO TRP             20      #             25      #             30ASN ALA SER GLY LYS LEU ILE         35 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 23 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:ASP GLN GLN LEU LEU GLY ILE TRP GLY CYS SE #R GLY LYS LEU ILE CYS1               5    #                10   #                15THR THR ALA VAL PRO TRP ASN             20(2) INFORMATION FOR SEQ ID NO: 8:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 27 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:ASN GLN GLN SER ARG TRP GLY LEU GLY SER PR #O ASN CYS HIS GLY PRO1               5    #                10   #                 15ASP TRP ALA SER PRO VAL CYS GLN ARG HIS SE #R             20      #            25 (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9:LYS ILE GLU PRO LEU GLY VAL ALA PRO THR LY #S ALA LYS ARG ARG VAL1               5    #                10   #                15VAL GLN ARG GLU LYS ARG              20

We claim:
 1. A compound of the formula:

wherein X is a C1 to C10 alkylene groups, an aromatic carbocyclic groupor a saturated carbocyclic group and R is the remainder of aregiospecific peptide having a terminal amino group and (i) free andinternal amino groups, (ii) free and internal hydroxyl groups, or (iii)free and internal amino and hydroxyl groups, wherein the compound issubstantially free of activated peptides comprising internal aminogroups, hydroxyl groups, or amino and hydroxyl groups bound to thecarbon atom of the amide carbonyl group; and further wherein R1 is theremainder of an enzyme characterized by the presence of a thiol groupbound thereby to the 3-position of the maleimide moiety, wherein thecompound is essentially homogeneous, the peptide being a salt of aprotonic acid.
 2. A compound according to claim 1 wherein X is anaromatic carbocyclic group.
 3. The compound according to claim 2 whereinX is phenyl.
 4. The compound according to claim 1 wherein R is theremainder of a peptide having a terminal amino group of the formula


5. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


6. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


7. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


8. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


9. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


10. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


11. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


12. The compound according to claim 1 wherein R is the remainder of apeptide having a terminal amino group of the formula


13. A compound according to claim 1 wherein R₁ is the remainder of aprotein characterized by the presence of a thiol group bound thereby tothe 3-position of the maleiimide moiety.
 14. A compound according toclaim 1 wherein the enzyme is thiolated.
 15. The compound according toclaim 14 wherein the thiolated enzyme is thiolated alkaline phosphatase.16. The compound according to claim 14 wherein the thiolated enzyme isβ-D-galactosidase.
 17. The compound according to claim 14 wherein thethiolated enzyme is thiolated peroxidase.
 18. The compound according toclaim 14 wherein the thiolated enzyme is thiolated glucose oxidase. 19.The compound of claim 1, wherein the protonic acid is a hydrohalic orhaloacetic acid.
 20. The compound of claim 19, wherein the hydrohalicacid is hydrobromic or hydrochloric acid.
 21. The compound of claim 19,wherein the haloacetic acid is trichioracetic or trifluoroacetic acid.